The present invention relates to a novel secretory cell function regulating protein and its DNA.
Regardless of prokaryotic or eukaryotic, cells secret various proteins through mechanisms inherent to them. In particular, a multicellular organism (living body) exchanges intercellularly a variety of signals to maintain its differentiation, proliferation and homeostasis, and various humoral factors that play pivotal roles for the signal transduction are mostly secretory proteins or mature proteins. These factors are classified into hormones, neurotransmitters, cytokines, growth factors, etc., based on the structural and functional properties. Through the advanced recombinant DNA technology and cell culture technology in recent years, it has been steadily proceeded to clarify genes encoding these proteins and their protein structures. On the other hand, discovery of such factors have made a breakthrough in analysis of receptors of these factors expressed on the cell surface and further have led to clarification of mechanism on intracellular signal transduction, which will characterize the physiological function of the cells. In many diseases in human or under pathological conditions of model animal with various diseases, it is often found that abnormal expression of some humoral factor that should normally maintain homeostasis causes these diseases, which, as a result, will lead to exacerbation. In addition, there is a phenomenon applicable to diagnosis of various diseases, such as a so-called tumor marker, in which the accentuated expression specifically observed in cancer, and its expression-controlling mechanism is also an important target in pharmaceutical discovery research.
MIA (melanoma inhibitor activity) reported by Blesch et al. in 1994 is one of the secretory proteins falling within such a category. At first, MIA was isolated from the culture supernatant of melanoma cells using an anti-proliferating activity against human melanoma cells as is seen from its name, and its gene was also acquired (Cancer Research, 54, 5695-5701, 1994). Subsequently in 1996, a homologous gene to this protein was again identified by Sandell et al. as CD-RAP (bovine cartilage-derived retinoic acid-sensitive protein), suggesting that CD-RAP will function to form and maintain the formation and maintenance of joints from a physiological aspect (The Journal of Biological Chemistry, 271, 3311-3316, 1996). Though the MIA/CD-RAP gene has a homology as high as 85% or more between species of human mouse, rat and bovine, any known homologous protein has not been found so far. From genetic analysis in bovine and rat, it was also considered that there was no other gene similar to the MIA/CD-RAP gene (The Journal of Biological Chemistry, 271, 3311-3316, 1996).
On the other hand, structural analysis of the full-length DNA one organism possesses, i.e., genome, has already been decoded in bacteria, and human genome analysis will also be completed in a few years. The predicted number of genes is said to reach 100,000 in human. Indeed, many genes encoding the secretory proteins or secretory peptides have been isolated so far, but on the whole, it cannot be said that even these many genes cover all of the entire genome. In understanding the phenomena of life on an individual level, intercellular signal transduction that would occur there must be all explainable. It is highly likely that some unknown humoral functional molecules other than such known genes may play physiologically critical roles, and it has been strongly desired to find such substances.
The present invention aims at providing a novel cell function regulating secretory protein (hereinafter sometimes referred to as MLP protein or MLP), its partial peptide or salts thereof, a DNA encoding the protein, a recombinant vector, a transformant, a method for manufacturing the protein, a pharmaceutical composition comprising the protein or the DNA, an antibody to the protein, a method and kit for screening a receptor agonist/antagonist, a receptor agonist/antagonist obtainable by the screening, as well as a pharmaceutical comprising the receptor agonist/antagonist, and the like.
Isolation of a novel cell function regulating secretory protein can not only lead to a new finding on the mechanism of differentiation, proliferation, malignant alteration, etc., but also can make a further progress to clarify the phenomena of life, including ontogenesis, maintenance of homeostasis, etc. and exhibit an inhibitory activity against or a promoting activity for the protein, resulting in development of a novel pharmaceutical useful for the prevention, diagnosis and treatment of various diseases.
The present inventors have made extensive studies and as a result, succeeded in cloning cDNAs each having a novel base sequence, from human fetal brain- and mouse fetal brain-derived cDNA libraries. The present inventors have found that proteins encoded by the thus obtained cDNAs are precursor proteins of MIA/CD-RAP-like protein MLP having a useful cell function regulating secretory activity and MLP formed after cleaving signal sequence out of the MLP precursor is a secretory protein. Based on these findings, the present invention have made further investigations and come to accomplish the present invention.
That is, the present invention provides the following features.
(1) A polypeptide containing an amino acid sequence, which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:24, its amide or ester, or a salt thereof.
(2) The polypeptide, its amide or ester, or a salt thereof, according to (1), which comprises an amino acid sequence that is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:6.
(3) The polypeptide or its amide or ester, or a salt thereof, according to (1), wherein substantially the same amino acid sequence represented by SEQ ID NO:24 is the amino acid sequence represented by SEQ ID NO:26.
(4) The polypeptide or its amide or ester, or a salt thereof, according to (2), wherein substantially the same amino acid sequence represented by SEQ ID NO:6 is the amino acid sequence represented by SEQ ID NO:12.
(5) The polypeptide or its amide or ester, or a salt thereof, according to (1), wherein substantially the same amino acid sequence represented by SEQ ID NO:24 is the amino acid sequence represented by SEQ ID NO:49.
(6) The polypeptide or its amide or ester, or a salt thereof, according to (2), wherein substantially the same amino acid sequence represented by SEQ ID NO:6 is the amino acid sequence represented by SEQ ID NO:47.
(7) A DNA containing a DNA bearing a base sequence encoding the polypeptide according to (1).
(8) The DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO:23.
(9) The DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO:4.
(10) The DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO:25.
(11) The DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO:10.
(12) The DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO:48.
(13) The DNA according to (6), wherein the base sequence encoding the polypeptide according to (1) is the base sequence represented by SEQ ID NO:46.
(14) A recombinant vector comprising the DNA according to (6).
(15) A transformant transformed with the recombinant vector according to claim 14.
(16) A method for manufacturing the polypeptide or its amide or ester, or a salt thereof, according to (1), which comprises culturing said transformant according to (15) and producing the polypeptide according to (1).
(17) An antibody to the polypeptide or its amide or ester, or a salt thereof, according to (1).
(18) A method of screening a compound or its salt that promotes or inhibits the activity of the polypeptide or its salt according to (1), which comprises using the polypeptide, its amide or ester, or a salt thereof, according to (1).
(19) A kit for screening a compound or its salt that promotes or inhibits the activity of the polypeptide, its amide or ester, or a salt thereof, according to (1), comprising the polypeptide or its salt according to (1).
(20) A compound or its salt that promotes or inhibits the activity of the polypeptide, its amide or ester, or a salt thereof, according to (1), which is obtainable using the screening method according to (18) or using the screening kit according to (19).
(21) A pharmaceutical comprising a compound or its salt that promotes or inhibits the activity of the polypeptide, its amide or ester, or a salt thereof, according to (1), which is obtainable using the screening method according to (18) or using the screening kit according to (19).
(22) A pharmaceutical comprising the polypeptide, its amide or ester, or a salt thereof, according to (1).
(23) An agent for the prevention/treatment of bone and joint diseases or pathologic angiogenesis, comprising the polypeptide, its amide or ester, or a salt thereof, according to (1).
(24) A diagnostic agent comprising the antibody according to (17).
The present invention further relates to the following features.
(25) The polypeptide, its amide or ester, or a salt thereof, according to (1), wherein substantially the same amino acid sequence as that represented by SEQ ID NO:24 is an amino acid sequence having homology of at least about 50% (preferably at least about 60%, more preferably at least about 70%, much more preferably at least about 80%, further much more preferably at least about 90%, and most preferably about 95%), to the amino acid sequence represented by SEQ ID NO:24.
(26) The polypeptide, its amide or ester, or a salt thereof, according to (1), wherein substantially the same amino acid sequence as the amino acid sequence shown by SEQ ID NO:24 is (i) an amino acid sequence represented by SEQ ID NO:24, of which 1 or 2 more (preferably approximately 1 to 30) amino acids are deleted; (ii) an amino acid sequence represented by SEQ ID NO:24, to which 1 or 2 more (preferably approximately 1 to 40, more preferably approximately 1 to 30) amino acids are added; (iii) an amino acid sequence represented by SEQ ID NO:24, in which 1 or 2 more (preferably approximately 1 to 30) amino acids are substituted by other amino acids; and (iv) a combination of the above amino acid sequences.
Furthermore, the DNA, polypeptide or its amide or ester or a salt thereof, etc. of the present invention can be utilized for basic studies, including molecular weight markers, tissue markers, chromosomal mapping, identification of hereditary diseases, design or primers or probes, etc.